The overall purpose of this proposal is to evaluate developmental nicotine-induced neurotoxicity. Despite the Surgeon General's warning concerning the harmful effects of smoking on the developing fetus, there is still a disturbing number of pregnant women who smoke during pregnancy. This proposal will utilize a rat model system to examine how and to what extent nicotine affects the developing brain. The proposal will test several hypotheses that are categorized into three Specific Aims. Specific Aim number 1 will test two hypotheses: 1) that nicotine exposure during all three trimesters equivalent will result in region-specific reductions in neuronal numbers in two important brain regions (hippocampus and cerebellum) in neonates and young adults; and 2) that the long-term brain deficits resulting from developmental nicotine exposure will be manifested through and correlated with specific behavioral impairments, spatial learning and parallel bars tasks, respectively. The exposure regimen used in Specific Aim number 1 is especially clinically relevant, since most pregnant women who smoke do so throughout pregnancy. Specific Aim number 2 will test two hypotheses: 1) that nicotine exposure during the third trimester equivalent (the brain growth spurt period) will lead to more severe neuronal loss than exposure restricted to first or first and second trimesters equivalent, and 2) that the cessation of nicotine exposure gestation will be beneficial to the developing brain. Specific Aim number 2 is important in addressing the questions regarding temporal vulnerability and the potential interaction between brain-regional specificity and temporal factors in mediating differential effects on nicotine-induced neuronal loss. Specific Aim number 3 will begin to address the question of mechanisms underlying nicotine-induced neuronal loss by testing the hypothesis that the application of specific neurotrophic factors (brain-derived neurotrophic factor [BDNF] and glial-derived neurotrophic factor [GDNF]) will attenuate nicotine-induced neuronal loss in an organotypic explant culture system. Specific Aim number 3 is the fist step to identify the involvement of specific neurotrophic factors as one of the underlying mechanisms for developmental nicotine-induced neuronal loss. The proposal will incorporate innovative in vivo and in vitro approaches to evaluate nicotine's toxicity during brain development, and many of the experimental techniques (artificial-rearing for third trimester equivalent exposure, 3-D stereological cell counting, organotypic explant culture system) proposed to be implemented in this proposal are novel to developmental nicotine research. The proposed studies will contribute to and broaden our knowledge of the harmful consequences from maternal smoking during pregnancy, provide a better understanding of the potential risk that may influence the severity of nicotine-induced brain deficits during different stages of development, and lead to a focus on mechanistic issues regarding developmental nicotine-induced neurotoxicity.